Monoclonal antibody DS6, tumor-associated antigen CA6, and methods of use thereof

ABSTRACT

The present application describes a monoclonal antibody selected from the group consisting of monoclonal antibody DS6, monoclonal antibodies that specifically bind to the antigen or epitope bound by monoclonal antibody DS6, and fragments of the foregoing that specifically bind to the antigen or epitope bound by monoclonal antibody DS6. Methods of use of such antibodies and the isolated antigen bound by such antibodies are also described.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/641,499, now issued U.S. Pat. No. 6,596,503, filed Aug. 18, 2000, thedisclosure of which is to be incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention concerns the monoclonal antibody DS6,tumor-associated antigen CA6, and methods of use thereof.

BACKGROUND OF THE INVENTION

Malignancy-associated changes of tumor cell membrane sialoglycoproteinsand sialoglycolipids (gangliosides) have been observed for decades andare implicated in many aspects of the transformed phenotype, includingaltered adhesive properties and metastatic potential (Bhavanandan, V.P., Furukawa, K., In: A. Rosenberg (ed.), Biology of the Sialic Acids,pp. 144–196, Plenum Press, New York (1995)). Altered, re-expressed orover-expressed sialoglycoconjugates, viewed as tumor-associated antigens(TAAs), are being investigated as targets for a variety ofantibody-directed cancer therapies. Some examples include monoclonalantibodies (mabs) directed against sTn (TAG-72) in breast cancer therapy(Estava, F. J. and Hayes, D. F. Monoclonal antibody-based therapy ofbreast cancer. In: M. L. Grossbard (ed.), Monoclonal antibody-basedtherapy of cancer, pp. 309–338, Marcel Dekker, New York (1998)) and mabsto gangliosides such as GD2, GD3 and GM2 in the therapy of lung cancerand melanoma (Butler, M. O. and Haluska, F. G., In: M. L. Grossbard(ed.), Monoclonal antibody-based therapy of cancer, pp. 339–364, MarcelDekker, New York (1998); Grant, S. C. et al., In: M. L. Grossbard (ed.),Monoclonal antibody-based therapy of cancer, pp. 365–395, Marcel Dekker,New York (1998)). Nevertheless, there remains a need for new ways todetect and treat cancer in patients.

SUMMARY OF THE INVENTION

The present invention is based upon the development of murine monoclonalantibody DS6 deposited in the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va., 20110-2209, on Jun. 11, 2002, anddesignated PTA-4449. This antibody immunohistochemically reacts with anantigen, CA6, that is expressed by human serous ovarian carcinomas butnot expressed by normal ovarian surface epithelium or mesothelium. TheCA6 antigen has a limited distribution in normal adult tissues and ismost characteristically detected in fallopian tube epithelium, innerurothelium and type 2 pneumocytes. Pretreatment of tissue sections witheither periodic acid or neuraminidase from Vibrio cholerae abolishesimmunoreactivity with DS6 indicating that CA6 is aneuraminidase-sensitive and periodic acid-sensitive sialic acidglycoconjugate (“sialoglycotope”). SDS-polyacrylamide gelelectrophoresis of OVCAR5 cell lysates reveals the DS6 epitope to beexpressed on an 80 kiloDalton nondisulfide-linked glycoproteincontaining N-linked oligosaccharides. Two-dimensional nonequilibrium pHgradient electrophoresis gels indicates an isoelectric point ofapproximately 6.2–6.5. DS6 immunostaining can be partially diminished bypretreatment of tissue sections with chloroform/methanol, suggestingthat DS6 may also be expressed as a glycolipid. Comparison of theimmunohistochemical distribution of the CA6 antigen in human serousovarian adenocarcinomas reveals similarities to that of CA125, however,distinct differences and some complementarity of antigen expression arerevealed by double-label, two-color immunohistochemical studies. TheDS6-detected CA6 antigen appears distinct from other well-characterizedtumor-associated antigens, including MUC1, CA125, and the histo-bloodgroup-related antigens, sLea, sLex and sTn.

A first aspect of the present invention is a monoclonal antibodyselected from the group consisting of monoclonal antibody DS6 andmonoclonal antibodies that specifically bind to the antigen or epitopebound by monoclonal antibody DS6, and fragments of the foregoing thatspecifically bind to the antigen or epitope bound by monoclonal antibodyDS6.

A second aspect of the present invention is a method of screening forthe presence of cancer in a human subject, comprising the steps of: (a)contacting a biological sample taken from said subject with an antibodyas described above under conditions permitting said antibody tospecifically bind an antigen in the sample to form an antibody-antigencomplex; and then (b) determining the amount of antibody-antigen complexin the sample as a measure of the amount of antigen in the sample,wherein an elevated level of the antigen in the sample is associatedwith the presence of cancer in said subject.

A further aspect of the present invention is a method of treating cancerin a subject in need thereof, comprising, administering to a subjectafflicted with cancer a monoclonal antibody as described above in atherapeutically effective amount.

A still further aspect of the present invention is isolatedtumor-associated antigen CA6, an about 80 kDa N-linked glycoprotein,reduced or non-reduced, with a PI of about 6.2–6.5, and containing asialiadase and periodate sensistive epitope called DS6. Such antigen maybe isolated by affinity purification with an monoclonal antibody DS6 asdescribed herein.

The foregoing and other objects and aspects of the present invention areexplained in detail in the drawings herein and the specification setforth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: DS6 immunohistochemical staining pattern on normal humantissues, A–D, with AEC (red) as the chromagen, hematoxylin counterstain:(A) Fallopian tube with apical staining of cells lining lumen, (B)apical aspect of type 2 pneumocytes of lung, (C) transitional epitheliumof ureter with DS6 staining of luminal-facing aspects of inner celllayers, (D) squamous metaplasia of uterine cervix with rim patternoutlining cell membrane.

FIG. 2: (A) DS6 immunoperoxidase buffer control, ie, withoutneuraminidase, on serous ovarian carcinoma, (B) DS6 immunoperoxidase onserous ovarian carcinoma following neuraminidase (Vibrio cholerae)treatment. C–F: Double label, sequential two color immunohistochemicalstaining of serous ovarian carcinomas with DS6 and OC125, hematoxylincounterstain: (C) DS6 (DAB, brown) followed by mouse Ig control (VIP,purple) shows a typical staining pattern for DS6 on tumors with bothluminal and cytoplasmic staining, (D) OC125 (DAB, brown) followed bymouse Ig control (VIP, purple) shows OC125 with a luminal pattern and afocus demonstrating a rim pattern along tumor cell membranes, (E) DS6(DAB) staining followed by OC125 (VIP) on a tumor demonstrating adiscreet area of staining (purple chromagen) that is DS6-nonreactive butOC125-reactive, (F) OC125 (DAB) staining followed by DS6 (VIP) stainingreveal areas of DS6-reactivity that were not detected by OC125.

FIG. 3: (A) Two-dimensional nonequilibrium pH gradient gelelectrophoresis (NEPHGE) analysis of the DS6-detected CA6 antigen.Samples were analyzed on NEPHGE gels (pI range ˜3.2–7.8) in the firstdimension, followed by 13% SDS-PAGE gels in the second dimension, allunder reducing conditions. (B) One dimensional SDS-PAGE analysis on 13%gels under reducing conditions and in (C) on 10% gels under nonreducingconditions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Any type of antibody may be used in the present invention. The term“antibodies” as used herein refers to all types of immunoglobulins,including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG areparticularly preferred. The antibodies may be monoclonal or polyclonal(with monoclonal antibodies preferred) and may be of any species oforigin, including (for example) mouse, rat, rabbit, horse, or human.See, e.g., M. Walker et al., Molec. Immunol. 26, 403–11 (1989). Antibodyfragments that retain specific binding to the protein or epitope boundby DS6 are included within the scope of the term “antibody” and include,for example, Fab, F(ab′)2, and Fc fragments, and the correspondingfragments obtained from antibodies other than IgG. Such fragments can beproduced by known techniques. The antibodies may be chimeric orhumanized, particularly when they are used for therapeutic purposes.

Subjects or patients with which the instant invention is concerned areprimarily human subjects, but the invention may also be employed withother mammalian subjects such as dogs, cats, and horses for veterinarypurposes. Subjects may be male or female.

Applicants specifically intend that all United States patent referencescited herein be incorporated herein by reference in their entirety.

1. Antibody Production.

Monoclonal antibodies of the present invention may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495–497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31–42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026–2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109–120). Briefly, the procedure is asfollows: an animal is immunized with antigen or immunogenic fragments orconjugates thereof. For example, haptenic oligopeptides of antigen canbe conjugated to a carrier protein to be used as an immunogen. Lymphoidcells (e.g. splenic lymphocytes) are then obtained from the immunizedanimal and fused with immortalizing cells (e.g. myeloma orheteromyeloma) to produce hybrid cells. The hybrid cells are screened toidentify those which produce the desired antibody.

Human hybridomas which secrete human antibody can be produced by theKohler and Milstein technique. Although human antibodies are especiallypreferred for treatment of human, in general, the generation of stablehuman-human hybridomas for long-term production of human monoclonalantibody can be difficult. Hybridoma production in rodents, especiallymouse, is a very well established procedure and thus, stable murinehybridomas provide an unlimited source of antibody of selectcharacteristics. As an alternative to human antibodies, the mouseantibodies can be converted to chimeric murine/human antibodies bygenetic engineering techniques. See V. T. Oi et al., Bio Techniques4(4):214–221 (1986); L. K. Sun et al., Hybricloma 5 (1986).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (S. L. Morrison, et al. Proc. Natl.Acad. Sci. 81, 6851–6855 (1984); M. S. Neuberger et al., Nature312:604–608 (1984); S. Takeda, S. et al., Nature 314:452–454 (1985)).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceCA6-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (D. R.Burton, Proc. Natl. Acad. Sci. 88,1120–3 (1991)).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature (R.Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833–3837 (1989)); G. Winteret al., Nature 349, 293–299 (1991)).

Polyclonal antibodies used to carry out the present invention may beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withthe antigen to which monoclonal antibody DS6 binds, collecting immuneserum from the animal, and separating the polyclonal antibodies from theimmune serum, in accordance with known procedures. Depending on the hostspecies, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund's,mineral gets such as aluminum hydroxide, and surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvantsused in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvumare especially preferable.

Antibodies that bind to the same epitope (i.e., the specific bindingsite) that is bound by the DS6 antibody can be identified in accordancewith known techniques, such as their ability to compete with labeledantibody to in binding to CA6 in a competitive binding assay.

Monoclonal antibodies specific for CA6 epitope can be used to produceanti-idiotypic (paratope-specific) antibodies. See e.g., McNamara etal., Science 220,1325–26 (1984), R. C. Kennedy, et al., Science 232,220(1986). These antibodies resemble the CA6 epitope and thus can be usedas an antigen to stimulate an immune response against CA6, or to screenother antibodies for the ability to specifically bind to the sameepitope bound by monoclonal antibody DS6.

DS6 can also be bound to a column (such as Protein A/G) and used toobtain purified CA6 antigen from a variety of sources, including humantissues/tumors and cancer cell lines that produce CA6. Such purified CA6antigen can then be used to produce additional antibodies (monoclonaland/or polyclonal) by methods described above. Some of these antibodiesmay react with the DS6 epitope while others can recognize differentepitopes on CA6. In one example, enzyme immunoassay to detect antigensin human body fluids often use a combination of antibodies thatrecognize different, non-sterically interfering epitopes on the sameantigen. For this purpose, a column containing immobilized neuraminidasecould be used to desialylate purified CA6; the desialylated CA6 can thenbe used as an immunogen to produce antibodies (monoclonal and/orpolyclonal) that react with non-DS6 epitopes on CA6. These antibodiescould then be used as either capture and/or tracer antibodies in anenzyme immunoassay for quantitation of the CA6 antigen for use inmonitoring of CA6 in pathologic states.

2. Immunoassay Techniques.

Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980)(CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for ModulatingLigand-Receptor interactions and their Application,” U.S. Pat. No.4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat.No. 4,376,110 to David et al., titled “Immunometric Assays UsingMonoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled“Macromolecular Environment Control in Specific Receptor Assays,” U.S.Pat. No. 4,233,402 to Maggio et al., titled “Reagents and MethodEmploying Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al.,titled “Heterogenous Specific Binding Assay Employing a Coenzyme asLabel.”

Antibodies as described herein may be coupled or conjugated to a solidsupport suitable for a diagnostic assay (e.g., beads, plates, slides orwells formed from materials such as latex or polystyrene) in accordancewith known techniques, such as precipitation. Antibodies as describedherein may likewise be conjugated to detectable groups such asradiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradishperoxidase, alkaline phosphatase), fluorescent labels (e.g.,fluorescein), chemiluminescent labels (e.g., acridinium groups,metalloporphyrins such as phthalocyanine dyes, luminol, etc.), metalatoms (e.g., technetium-99 m), etc., in accordance with knowntechniques. See, e.g., U.S. Pat. No. 4,472,509 to Gansow (metal chelatesto monoclonal antibodies); U.S. Pat. No. 5,061,641 to Schochat et al.;and U.S. Pat. No. 4,861,869 to Nicoleotti et al. (radiolabellingproteins).

Immunoassays, or other types of assays to detect and/or quantitate thelevel of the CA6 antigen in samples as described below, may be used inscreening assays to detect pathologic states associated with aberrantlevels of CA6 expression (e.g., tumors, inflammatory states), diagnosticstudies, prognostic studies, or to monitor the progression or diminutionof CA6 expression in correlation with disease state.

Samples that may be collected for use in carrying out the immunoassaymay be tissue samples from the organ or tissue of interest within thesubject, such tissue generally of most interest being those types oftissues/cells that express differing amounts of CA6 in pathologic statesas compared to non-pathologic states, or biological fluids such as blood(including blood fractions such as blood plasma or blood serum), urine,cerebrospinal fluid, etc). Examples may include overexpression oraberrant expression of CA6 in various types of malignancies as will beseen in the Tables below (e.g ovarian cancer, endometrial cancer,pancreatic cancer, breast cancer, urinary bladder cancer, lung cancer,etc.), as well as overexpression or aberrant expression in otherpathologic states, such as overexpression of CA6 by pneumocytes in lungsdisease, for example, pneumonia.

A biological sample may be a cell sample, with an intervening culturingstep being performed between the time the cell sample is collected fromthe subject and the immunoassay is carried out on the biological sample.

For immunohistological techniques, a tissue sample is collected from thesubject, and the presence or absence of binding of an antibody of theinvention is detected. The presence of binding of the antibody in anabnormal pattern or a pattern indicative of a tumor or cancer indicatesthe presence of a tumor or cancer in the subject from which the tissuesample is collected. The presence of the antigen in a metastatic tumordeposit can also be used to determine a likely source of the primarytumor. Any suitable immunohistology format may be used. The tissuesample may include patient biopsies, resections or cells for cytologicstudy. A similar technique to immunohistology is the use of similartechniques to detect and/or phenotype cells in body fluids or othersuspensions as is used for flow cytometric examination.

For in vivo diagnostic purposes the antibody according to the inventionis coupled to or provided with a suitable externally detectable label,such as e.g. a radiolabel as described above or a metal atom (e.g.,technetium-99 m), and administered to a subject (e.g., by intraveneousor intraarterial injection), in an amount sufficient to produce anexternally detectable signal, whereupon the possible localizedaccumulation of antibody in the body is determined, with a localizedaccumulation of the antibody (in a region other than that which wouldordinarily be expected for normal subjects or subjects free of disease)indicating the present of a tumor in that subject.

3. Therapeutic Antibodies and Methods.

Monoclonal antibodies used for therapy (i.e., in a method of combattingcancer) may be monoclonal antibodies per se or monoclonal antibodiescoupled to a therapeutic agent. Such antibodies are referred to hereinas therapeutic monoclonal antibodies. Any therapeutic agentconventionally coupled to a monoclonal antibody may be employed,including (but not limited to) radioisotopes, cytotoxic agents, andchemotherapeutic agents (See generally Monoclonal Antibodies and CancerTherapy (R. Reisfeld and S. Sell Eds. 1985)(Alan R. Liss Inc. NY); U.S.Pat. No. 5,558,852 to Bigner and Zalutsky; U.S. Pat. No. 5,624,659 toBigner and Zalutsky).

Therapeutic agents may be conjugated or coupled to the antibody bydirect means or indirect means (e.g., via a chelator), such as theIodogen method or with N-succinimidyl-3-(tri-n-butylstanyl)benzoate (the“ATE method”), as will be apparent to those skilled in the art. See,e.g., M. Zalutsky and A. Narula, Appl. Radiat. Isot. 38, 1051 (1987).

Examples of radioisotopes which may be coupled to a therapeuticmonoclonal antibody include, but are not limited to, ¹³¹I, ⁹⁰Y, ²¹¹At,²¹²Bi, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, and ²¹²Pb. Examples of chemotherapeuticagents which may be coupled to a therapeutic monoclonal antibodyinclude, but are not limited to, methotrexate. Examples of cytotoxicagents which may be coupled to a therapeutic monoclonal antibodyinclude, but are not limited to, ricin (or more particularly the ricin Achain).

The monoclonal antibodies of the invention can be conjugated to and usedas targeting agents for genes (immunogenes, suicide genes),immunoliposomes, boron neutron capture therapy, photosensitizers forphotodynamic therapy, and other types of therapies that can be directedby antibodies.

It will be appreciated that monoclonal antibodies per se which are usedas therapeutic monoclonal antibodies incorporate those portions of theconstant region of an antibody necessary to evoke a therapeuticallyuseful immunological response in the subject being treated.

Therapeutic monoclonal antibodies may be provided in lyophylized form ina sterile aseptic container or may be provided in a pharmaceuticalformulation in combination with a pharmaceutically acceptable carrier,such as sterile pyrogen-free water or sterile pyrogen-free physiologicalsaline solution.

The therapeutic methods disclosed herein may be employed with subjectssuspected of having a variety of tumors, whether primary or metastaticor micrometastatic (see Tables below), of particular importance aretumors of the ovary, endometrium, breast, urinary bladder, pancreas andlung. DS6 may also be of therapeutic use in other types of neoplasms,especially if used as part of a panel or combination of therapeuticantibodies, each with different specificities (Smith, N. L. et al, HumanAntibodies, 9, 61–65, (1999); Oldham, R. K., Mol Biother., 3, 148–161,(1991)).

For administration, the antibody will generally be mixed, prior toadministration, with a non-toxic, pharmaceutically acceptable carriersubstance (e.g. normal saline or phosphate-buffered saline), and may beadministered using any medically appropriate procedure, e.g.,intravenous or intra-arterial administration, injection into thecerebrospinal fluid). In certain cases, intradermal, intracavity,intrathecal or direct administration to the tumor or to an arterysupplying the tumor is advantageous.

Dosage of the antibody will depend, among other things, on the tumorbeing treated, the route of administration, the nature of thetherapeutic agent employed, and the sensitivity of the tumor to theparticular therapeutic agent. For example, the dosage will typically beabout 1 to 10 micrograms per Kilogram subject body weight. In anotherexample, where the therapeutic agent is ¹³¹I, the dosage to the patientwill typically be from 10 mCi to 100, 300 or even 500 mCi. Statedotherwise, where the therapeutic agent is ¹³¹I, the dosage to thepatient will typically be from 5,000 Rads to 100,000 Rads (preferably atleast 13,000 Rads, or even at least 50,000 Rads). Doses for otherradionuclides are typically selected so that the tumoricidal dose willbe equivalent to the foregoing range for ¹³¹I. The antibody can beadministered to the subject in a series of more than one administration,and regular periodic administration will sometimes be required.

The present invention is explained in greater detail in the followingnon-limiting Examples. Abbreviations used: mab (monoclonal antibody),TAA (tumor-associated antigen), IH(immunohistochemistry/immunohistochemical), kiloDalton (kDa).

EXAMPLE 1 Monoclonal Antibody DS6 Detects a Tumor-AssociatedSialoglycotope Expressed on Human Serous Ovarian Carcinomas

We have recently developed a murine monoclonal antibody (mab), cloneDS6, using human serous ovarian carcinoma as the immunogen (Smith, N. L.et al., Human Antibodies, 9, 61–65, (1999); Kearse, K. P. et al, Int. J.Cancer, in press, (2000)). We now report the immunohistochemical (IH)distribution of the DS6-detected antigen, CA6, in normal adult humantissues and selected neoplasms and characterize the epitope as asialoglycotope.

1. Materials and Methods

Hybridoma production. Immunizations, fusions and screening wereperformed essentially as described previously using P3X63-Ag8.653myeloma cells with human serous ovarian carcinoma as the immunogen(Wennerberg, A. E. et al., Am. J. Pathol., 143(4), 1050–1054 (1993)).Institutional Animal Care and Use approval was obtained. Preliminary andsecondary screenings were by avidin-biotin IH (mouse IgG peroxidase kit,Vector Laboratories, Burlingame, Calif.) on tissue sections of ovariancarcinomas and selected normal adult tissues. DS6 was chosen for furtherstudies and, after several rounds of single cell cloning by limitingdilution, was isotyped as an IgG1 (ImmunoType Kit, Sigma Chemicals, St.Louis, Mo.). Supernatant was collected in batch for the studies in thisreport from DS6 cells grown in DMEM-F12 supplemented with 10% horseserum. Quantitation of murine IgG1 in the supernatant was by EIAmethodology (performed by the East Carolina University Hybridoma CoreFacility, Greenville, N.C.).

Immunohistochemistry. Tissue culture supernatant (40 ug DS6/ml) was usedfor IH as above with AEC (Vector Laboratories) as the chromagen. Frozencryostat tissue sections were air-dried, acetone-permeabilized whileformalin-fixed, paraffin-embedded tissue sections were de-waxed throughsolvents and rehydrated, prior to blocking of endogenous peroxidase withmethanol/hydrogen peroxide and subsequent immunostaining. Mouse IgG1 wasused as a negative control mab. Anti-transferrin receptor mab (IgG1) wasused as a positive control on frozen cryostat sections while anti-smoothmuscle myosin mab (IgG1) was used as a positive control onformalin-fixed tissues (all control antibodies from DAKO, Carpenteria,Calif.).

Human adult tissues. Samples of grossly normal, incidental tissues wereobtained fresh from either autopsy or the surgical bench fromindividuals without significant pathology in the primary organ ortissue; half were used for air-dried, acetone-permeabilized cryostatsections and half were fixed in buffered formalin and paraffin-embedded.Additional normal tissues as well as a pilot selection of gynecologicand related neoplasms were obtained from the archival tissue stores offormalin-fixed, paraffin-embedded tissue blocks of the Universityhospital (Table 1 and Table 2). Fresh specimens of serous ovarian tumorsand normal fallopian tubes were obtained from surgical cases andcryostat sections were cut for chloroform extraction studies. Use ofincidental human tissues had prior approval of the Institutional ReviewBoard.

Following completion of the normal tissue distribution and pilot studyon gynecologic tumors, an expanded study was undertaken. 296formalin-fixed, paraffin-embedded human gynecologic neoplasms wereimmunohistochemically examined using DS6 as the primary antibody (Table3). A large study of non-gynecologic neoplasms followed (n=approx 1200)as seen in Tables 4–8.

Pretreated and/or preabsorbed immunohistochemical studies. For periodicoxidation of carbohydrates, formalin-fixed, paraffin-embedded sectionsof normal human stomach, small bowel, colon, fallopian tube, breast andserous ovarian carcinoma were pretreated, prior to IH, with 20 mMperiodic acid in 0.5 M acetate buffer, pH 5.0 as described by Cao (Cao,Y. et al., J. Histochem. & Cytochem., 45(11), 1547–1557 (1997); Cao, Y.et al., Tumor Biol. 19(1), 88–99 (1998).). Parallel control sectionswere incubated in acetate buffer without periodic acid to control forloss of reactivity due to pH or buffer. For detection of sialic acid,0.02 U/ml neuraminidase from Vibrio cholerae (reacts with sialic acidsin α 2–3,-6,-8 linkages, Boehringer Mannheim/Roche, Indianapolis, Ind.),in 0.01M Ca++in PBS was used to pretreat formalin-fixed tissue sectionsof the above tissues with parallel control sections pretreated withCa++/PBS buffer without neuraminidase. An additional control for theadequacy of sialic acid removal (via unmasking of PNA lectin-bindingsites) was the inclusion of a section of formalin-fixed kidney, run asabove, but stained with PNA-biotin (EY Labs, San Mateo, Calif.) in placeof the DS6 primary and biotin-labeled secondary antibodies. For removalof glycolipids prior to DS6 immunostaining, unfixed, air-dried cryostatsections of normal fallopian tubes and serous ovarian tumors were placedin 5% chloroform in methanol for 10 minute (Zhang, S. et al., Int. J.Cancer, 73, 50–56 (1997)).

To determine if DS6 reacts with several commercially available, highlypurified carbohydrate antigens (sTn, Tn, Tf, sLea, Calbiochem, La Jolla,Calif.), the antigens were used to preabsorb DS6 prior to immunostaining(Taylor, C. R., In: C. R. Taylor (ed.), Immunomicroscopy. A diagnostictool for the surgical pathologist, pp. 23–43, W. B. Saunders,Phildelphia (1986); Elias, J. M., Immunohistopathology: a practicalapproach to diagnosis, pp. 53–56, American Society of ClinicalPathologists, USA (1990)). Briefly, carbohydrate antigens were added toDS6 supernatant at 10 ug, 50 ug, 250 ug and 500 ug carbohydrate to 4 ugDS6 and allowed to react at 4° C. overnight. This preabsorbedsupernatant was then used by IH to study sections of formalin-fixed,paraffin-embedded serous ovarian carcinomas and compared to controlsections stained with unabsorbed DS6 supernatant.

Double-immunolabel, two-color IH on tissue sections. Small sections (upto 1.0 cm) of eight formalin-fixed and paraffin-embedded serousadenocarcinomas and a metastasis of each were re-embedded into twoparaffin blocks for single antibody and double label/sequential IH(Battifora, H., Lab. Invest., 55, 244–248 (1986)). Seven of the cases ofadenocarcinoma had their primary site in the ovary, and one case was aprimary papillary serous carcinoma of the peritoneum. A section ofnormal fallopian tube was also included in each block. A manufacturer'sprotocol (Vector Laboratories) for two-color, double-label IH was used:in brief, one of the murine mabs is used in a standard avidin-biotin IHtechnique with DAB (brown product, Vector Laboratories) as chromagen andthen the entire process is repeated on the same slide using thealternate murine mab with VIP (“Very Intense Purple”; purple product,Vector Laboratories) as the chromagen. DS6 (neat) supernatant was usedas one mab and a murine anti-Ca125 (OCH125 Level I, an IgG1, SignetLaboratories, Dedham, Mass.) as the alternative mab. DAB wasconsistently used as the first chromagen and VIP as the second chromagenin the sequence. For double-label studies with both DS6 and OC125, theorder of the primary antibodies was switched while maintaining the orderof the chromagens so that each mab was evaluated both as a first andsecond mab in the sequence. Controls consisted of a primary mab (DS6 orOC125) with DAB followed by a mouse ascites negative control (SignetLaboratories) as the second mab with VIP. Antigen retrieval (10 mM, pH6.0 citrate buffer) by microwave proceeded all double-label staining toassure antigen detection by mab OC125.

Immunoblotting and SDS-PAGE gel electrophoresis. Lysates of OVCAR5, ahuman ovarian carcinoma cell line (a generous gift from Dr. ThomasHamilton, Fox Chase Cancer Center, Philadelphia, Pa.) that is reactivewith DS6 by IH, were analyzed on one and two-dimensionalSDS-polyacrylamide gel electrophoresis as previously described (VanLeeuwen, J. E. M. and Kearse, K. P., J. Biol. Chem., 271, 25345–25349(1996)). Gels were transferred to a nitrocellulose membrane and probedwith DS6 or control antisera diluted in PBS containing 5% powdered milk,washed, and incubated with protein A-HRP (ICN, Irvine, Calif.) in PBS(5% milk). Protein A-HRP complexes were visualized by chemiluminescenceperformed according to manufacturer's instructions (Pierce ChemicalCompany, Rockford, Ill.). N-linked glycans were removed fromprecipitated proteins using an Enzymatic Deglycosylation kit (Glyko,Inc., Novato, Calif., USA).

2. Results

Distribution of CA6 in normal tissues and tumors by IH. The results ofIH staining using DS6 on formalin-fixed, paraffin-embedded tissues aresimilar to those obtained on acetone-permeabilized cryostat sections,consistent with an epitope that is fairly resistant to the effects offormalin-fixation. The IH pattern of DS6 on cells of normal adulttissues is predominately an apical, epithelial cell membranelocalization in cells lining certain tubular or saccular structures(e.g., fallopian tube, pulmonary alveoli) and along the luminal-facingaspect of the inner layers of some stratified epithelia, such asurothelium (FIG. 1, A–C). In a few instances, such as metaplasticcervical epithelium and squamous metaplasia of amnion, the entire cellmembrane can be decorated giving a rim pattern to the cell (FIG. 1, D).There is absent to minimal cytoplasmic expression of the antigen in anyof the normal human adult epithelia studied.

The immunohistochemical distribution of the CA6 antigen in various typesof normal adult tissues in given in Table 1. In normal adult tissues,the CA6 antigen is most consistently detected in fallopian tubeepithelium, urothelium and type 2 pneumocytes. In other tissues thathave DS6 immunoreactivity, the CA6 antigen has a more inhomogeneous andvariable expression pattern. This is particularly evident in ductalstructures, where expression can vary from one duct cross section toanother within a given histologic preparation.

In a pilot study of selected gynecologic neoplasms (Tables 2), CA6 isexpressed along the apical membranes of many cells in ovarian serouscystadenomas. In ovarian serous adenocarcinomas, the level of CA6expression is more intense and is additionally seen in the cytoplasm ofmany malignant cells and extracellularly within gland lumen (FIG. 2, C).A similar luminal/apical pattern with variable cytoplasmic staining isalso seen in the other adenocarcinomas that are DS6 immunoreactive.Mucinous ovarian tumors are not characteristically DS6 immunoreactive asno reactivity is seen in benign and borderline mucinous tumors and isonly seen quite focally in 3 of 9 mucinous adenocarcinomas. CA6 is notdetected in normal or hyperplastic mesothelium and does not appear to becharacteristic of mesotheliomas (1/6), but is readily detected in seroussurface carcinomas of the peritoneum (3/3).

Sensitivity of CA6 epitope to periodic acid, neuraminiclase, andchloroform extraction. To characterize the CA6 epitope, sections ofDS6-reactive (IH) fallopian tube and serous ovarian carcinoma, as wellas DS6 nonreactive stomach, small bowel, colon and breast, weresubjected to a series of pretreatments with either periodic acid orneuraminidase (V. cholera) prior to immuonostaining with DS6.Pretreatment of the tissue sections with either periodic acid orneuraminidase (sialidase) prior to IH with DS6 results in completeabolishment of DS6 immunoreactivity in the sections of fallopian tubeand ovarian carcinoma and no DS6 immunoreactivity was unmasked in any ofthe sections of gastrointestinal tissues or breast. DS6 reactivityremained unaffected in the buffer control sections (FIG. 2, A–B).Preabsorption of DS6 supernatant with commercially available purifiedcarbohydrate antigens (sTn, sLea, Tf, Tn) under conditions of antigenexcess failed to reduce the intensity of DS6 immunostaining of serousovarian carcinomas (data not shown).

Chloroform extraction studies were performed on sections of fallopiantubes (2) and serous ovarian tumors (3) prior to immunostaining withDS6. Control cryostat sections of fallopian tubes and ovarian tumorsshow strong staining with DS6 in the formalin-fixed as well as in theacetone-permeabilized slides. The chloroform-pretreated slides, stainedwith DS6, reveal mild to moderate reduction in staining intensity infallopian tubes and ovarian tumors respectively.

Immunoblotting and SDS-polyacrylamide gel electrophoresis. OVCAR5, ahuman ovarian carcinoma cell line reactive with DS6 by IH (data notshown) was used as a reproducible source of antigen. Anti-DS6immunoblots of OVCAR5 lysates gave a predominant band at approximately80 kilodaltons (kDa), with minor species at 75 kDa, 52 kDa, and 48 kDa(FIG. 3, B). Migration is similar under both reducing and nonreducingconditions and no reactivity with control (isotype matched) antibody isobserved (FIG. 3, B–C). Analysis on two-dimensional nonequilibrium pHgradient electrophoresis (NEPHGE) gels indicates an isoelectric point ofapproximately 6.2–6.5 (FIG. 3, A). Digestion studies with PNGase Fresult in a reduction in molecular weight of the 80 kDa species toapproximately 75 kDa, consistent with the presence of one to two N-likedglycan chains (data not shown).

Double-immunolabel, two-color IH of tissue sections. Single antibody anddouble-label, two-color IH studies were performed using mabs DS6 andOC125 to determine if CA6 and CA125 are expressed by the same ordiffering tumor cell populations. Both antibodies reacted with theluminal aspect of fallopian tubes but only OC125 reacted with themesothelium of the fallopian tubes. All eight primary papillary serousadenocarcinomas and their metastases show moderate to strong positivityfor DS6 and OC125 with the exception of one primary tumor that has onlyscattered focal positive cells with either mab. Comparison of tumorsstained individually with either DS6 or OC125 reveal that they reactwith roughly similar proportions of tumor cells although the intensityof staining is more pronounced with DS6. While both the CA6 and CA125antigens are expressed along the luminal membranes of the neoplasticcells in many areas and in most tumors strikingly similar IH patternscan be found, there are some distinct differences. CA6 often has anadditional prominent cytoplasmic component while CA125 will occasionallyshow a distinct pattern of circumferential membrane expression on theneoplastic cells in several of the tumors, giving a honeycomb appearanceto the tumor that is not as characteristic of CA6 (FIG. 2, C–D). Whenboth DS6 and OC125 are used in sequential, two-color IH, intenselypositive dark chromagen results from overlapping, dual localization ofchromagens in many areas of the tumors. This dark chromagen can bedifficult to distinguish from an intensely positive deposition from thefirst mab/chromagen (brown, DAB) reaction alone. However, in mosttumors, focal areas of chromagen deposition resulting from the secondmab/chromagen (purple, VIP) reaction can be clearly visualized (FIG. 2,E–F). Such areas result from the second mab in the sequence reactingwith an antigen that is not detected by the prior application of thefirst mab.

Following the immunohistochemical studies above, a greatly expandednumber of gynecologic neoplasms, as well as non-gynecologic neoplasms,were similarily studies for DS6 immunoreactivity. The results are listedin Tables 3–8. As can be seen, the CA6 antigen is not restricted toovarian carcinomas but can be expressed by a variety of othercarcinomas, including, but not limited to, those of the breast,endometrium, pancreas, urinary bladder and lung.

3. Discussion

As CA6 has only a limited distribution in the normal tissues studied, isnot detected in normal ovarian surface epithelium, yet is expressed inserous ovarian carcinomas which arise from the surface epithelium(18/18), it can be described as a TAA (Suresh, M. R., Anticancer Res.,16(4B), 2273–2277 (1996); Khawli, L. A. and Epstein, A. L., Q.J. Nucl.Med., 41, 25–35 (1997)). Immunohistochemical studies of DS6 on a widevariety of human carcinomas, lymphomas and sarcomas can be seen inTables 2–8 and demonstrate that DS6 is not a pan-carcinoma marker butrather is characteristic of specific types of epithelial malignancies(e.g., characteristic of serous ovarian carcinomas but seldom reactivewith colon carcinomas). Analyzing the IH distribution of CA6 in normaltissues, similarities, as well as differences, can be noted to otherreported TAAs. For instance, the pattern of reactivity in the anteriorpituitary has also been described for CA19-9 and EMA (Okubo, T. andOkabe, H., Acta Neuropathol., 93, 471–476 (1997); Pernicone, P. J. etal., In: S. S. Sternberg (ed.), Histology for pathologists, secondedition, pp 1053–1074, Lippincott-Raven, Philadelphia (1997)), thestaining of the intracellular canaliculus of parietal cells is a featureof the MUC1-related mab HMFG2 (Walker, M. M. et al., J. Clin. Pathol.,48, 832–834 (1995)), while luminal staining of ductal structures isvariably reported for a large number of TAAs, including the MUC1 mucinfamily and histo-blood group related antigens (Arklie, J. et al., Int.J. Cancer, 28, 23–29 (1981); Zhang et al., supra (1997); Cao, Y. et al.,Histochem. Cell Biol., 106, 197–207 (1996)). Reports of mabs to TAAshave been numerous and, as TAAs are not unique to tumors, it is notunexpected that some similarities in their IH distribution in normalepithelial tissues would be seen. The reported distribution ofepithelial TAAs can also vary in the hands of different investigators,perhaps reflecting differences in methodologies, grading systems,antibody preparation, tissue preparation, or fixation (Zhang et al.,supra (1997); Stein, R. et al., Int. J. Cancer, 47,163–169 (1991);Buist, M. R. et al., J. Clin. Pathol., 48, 631–636 (1995)). We havecharacterized the IH distribution of DS6 in both cryostat andformalin-fixed, paraffin-embedded tissues to provide a range ofexperimental conditions that are in common use. Additionally, we haveclassified the types of DS6-reactive epithelial tissues as to the typesof tissues most consistently positive versus those tissues in which theexpression appears more variable. As will be discussed below, theconsistent presence or absence of CA6 in certain epithelial tissues doesappear to distinguish CA6 from certain other known TAAs. To furthercharacterize CA6, additional features of the antigen were investigated.

Pretreatment of tissue sections with either periodic acid orneuraminidase from Vibrio cholerae gives similar results. Thesetechniques are used to determine if the epitope is carbohydrate-based orsialic acid-dependent, respectively, and have also been used to unmaskhidden epitopes such as masking of the binding sites of several MUC1mabs by glycosylation (Cao et al., supra (1997); Cao et al, supra(1998)). The abolishment of DS6 immunoreactivity by both treatments isconsistent with a carbohydrate epitope that is sialic acid-dependent. NoDS6-reactive epitopes are unmasked in any of the sections by suchremoval of periodic-sensitive carbohydrates or sialic acid.

To determine if the CA6 sialoglycotope is expressed as asialoglycoprotein and/or a sialoglycolipid (ganglioside), additionalstudies were performed. SDS-PAGE immunoblots reveal that the DS6 mabdetects a predominant 80 kDa glycoprotein with N-linkages. Minor sidebands are noted that may represent proteolytic degradations of the majorband or are side reactivities of the antibody (ie, carbohydrate epitopeshared on more than one protein). No change in migration of the major 80kDa band is noted between reduced and non-reduced conditions signifyinga lack of interchain disulfide bonds (FIG. 3, B–C). Chloroformextraction studies are used to extract lipids from tissue sections priorto DS6 immunostaining and are then compared to DS6 immunostaining ontissue sections without prior chloroform extraction. While some generalreduction in staining intensity was noted in sections of fallopian tube,a more marked reduction was noted in the sections of ovarian tumors.Whether this apparent difference in efficiency of extraction in a normaltissue (i.e., fallopian tube) as compared to tumors is quantitative orqualitative is uncertain, however, ongoing studies into the chemicalnature of the epitope may help explain the significance of this finding.The sialoglycotope nature of CA6 is similar to many of the histo-bloodgroup-related antigens such as sTn, sLea, sLex, some of which aredually-expressed as glycoproteins and glycolipids, however, unlike CA6,as glycoproteins, these are usually high molecular weight, carbohydraterich O-linked glycoproteins (mucins) (Magnani, J. L. et al., CancerRes., 43, 5489–5492 (1983); Muraro, R. et al., Cancer Res., 48,4588–4596 (1988)). The immunohistochemical distribution of thehisto-blood group related antigens, as reviewed by Zhang et al., wouldalso distinguish CA6 from the above sialyated histo-blood group antigens(Zhang et al., supra (1997)). The apical-staining pattern of DS6 on type2 pneumocytes, which lack sTn, sLea and sLex expression (Hachiya, T. etal., Virchows Arch., 434, 63–69 (1999); Atkinson, B. F. et al., CancerRes., 42, 4820–4823 (1982)), is strikingly similar to that reported forthe sTf (sialyl-Thomsen-Fridenreich) antigen (Hachiya et al., supra(1999)). However, if DS6 is reacting with the sTf epitope per se, itwould be expected to demonstrate a much broader range ofimmunoreactivity in human tissues than is seen in our studies, includingexpression in DS6-nonreactive epithelia, brain, lymphoid and othermesenchymal tissues (Cao et al., supra (1996)). The DS6 preabsorptionstudies using commercially available, highly purified sTn, sLea, Tn andTf carbohydrate antigens, in which the antigens failed to inhibit thetissue binding of DS6, also support CA6 being distinct from thesecarbohydrate antigens. Other TAAs to consider are those of approximately80 kDa such as OC133 (Berkowitz, R. et al., Am. J. Obstet. Gynecol.,146(6), 607–612 (1983); Masuho, Y. et al., Cancer Res., 44, 2813–2819(1984)), Ki-OCI-6-2 (Mettler, D. L. et al., Cancer, 65, 1525–1532(1990)), and the lactoferrin-related MAM5 (Zotter, S. et al., VirchowsArch. A. Pathol. Anat. Histopathol., 406(2), 237–251 (1985)), none ofwhich are expressed by fallopian tube, and 22-1-1, a mab to aneuraminidase resistant epitope on a 78 kDa protein, which is morecharacteristic of mucinous rather than serous ovarian tumors (Sonoda, K.et al., Cancer, 77(8), 1501–1509 (1996)).

Although the molecular weights of CA6 (80 kDa) and CA125 (>200 kDa) andtheir distribution in normal tissues differ (CA125 has been detected byIH in some normal epithelia of pancreas, ovary, fallopian tube, colon,gallbladder, stomach, endometrium, bronchus, lung, kidney and inmesothelium and amnion), the IH pattern of CA6 in our series of ovariantumors is similar to that reported for CA125 (Davis, H. M. et al.,Cancer Res., 46, 6143–6148, 1986; Nouwen, E. J. et al., Cancer Res.,46(2), 866–876 (1986); Dietel, M. et al., J. Cancer Res. Clin. Oncol.,111, 257–256 (1986); Itahashi, K. et al., Arch. Gyn. Obst., 243(3),145–155 (1988)). Both antigens are more characteristic of ovarian serouscarcinomas than ovarian mucinous neoplasms and both have a variegateddistribution pattern, often with luminal accentuation (Mattes, M. etal., Cancer Res. (suppl.), 50, 880–884 (1990)). In IH studies, bothantigens show a spectrum of expression ranging from strong and diffusein some tumors to patchy and variegated patterns in other neoplasms,with strongly positive groups of cells adjacent to nonreactive foci. Thedouble-label, two-color, sequential IH studies comparing CA6 to CA125demonstrate that mabs DS6 and OC125, while showing extensive overlap,also show areas in which only one of the antibodies is expressed,providing additional, complementary coverage of the tumor. Suchcomplementarity of TAA expression can have important clinicalimplications, whether for tailoring mab-based cancer therapies as“cocktails” matched to a given tumor's antigenic phenotype (Liao, S.-K.et al., Cancer Immunol. Immunother., 28, 77–86 (1989); Oldham, R. K.,Mol. Biother., 3, 148–161 (1991)), or for devising panels of tumormarkers to monitor tumor progression (Cane, P. et al., GynecologicOncol., 57, 240–245 (1995)).

In the sections of fallopian tubes, CA125 is distributed along luminalepithelial surfaces and is readily detected in the mesothelium.Conversely, CA6, which is more intensely expressed along the fallopiantube lumen, is not detected in the mesothelium. DS6 clearly recognizesan antigen that has a similar, but not identical, distribution to theCA125 antigen, with some distinct differences in patterns and sites ofantigen expression. The lack of CA6 expression in normal mesotheliumcould make DS6 a candidate for intraperitoneal antibody-targetedtherapeutic applications.

TABLE 1 Immunohistochemical detection of CA6 in tissue sections ofnormal adult tissues. Acetone-permeabilized and formalin-fixed,paraffin-embedded tissues were stained with mab DS6 as the primaryantibody by avidin-biotin immunohistochemistry. CryostatFormalin-paraffin #positive/ #positive/ Tissue #tested #tested Positivecell types/Comments Tissues with prominent, consistent CA6 expressionwithin and between specimens: Fallopian tube 3/3 6/6 Luminal/apicalepithelial cell membrane  (8/8)^(a) Lung 2/2 5/5 Apical-aspect of typeII pneumocyte; occasional epithelial cell of bronchiole Urothelium 3/38/8 Inner layers, luminal-facing aspect Tissues with low-level CA6expression, quite variable/focal within and between specimens: Pancreas2/2 5/6 Luminal-aspect of small to medium ducts Liver 1/2 3/5Luminal-aspect of occasional portal bile ducts Major salivary gland nt^(b) 2/3 Luminal-aspect of occasional medium-sized ducts Kidney 3/35/5 Luminal-aspect of scattered cells of distal nephron Endometrium,secretory 3/3 3/4 Focal luminal staining Rare surface epithelial cell,2/4 Cervix 2/2 5/6 Focal, membrane rim pattern in metaplasticepithelium, transformation zone and 1/1 ectocervical epitheliumEndocervical glands  0/2^(c) 3/6 Focal luminal staining of some glandsin transformation zone Pituitary 2/2 2/2 Focal staining of rim offollicles, anterior lobe Tissues lacking CA6 expression or rare focusonly: Cerebral cortex 0/3 0/3 Cerebellum 0/3 0/3 Spinal cord 0/3 0/3Large peripheral nerve 0/3 0/3 Trachea nt 0/2 Parathyroid 0/2 1/4 Onesmall focus of CA6 in histologically normal area of gland that elsewherehad pseudofollicle formation Thyroid 2/2 2/3 Rare apical-aspect offollicular epithelial cell Breast 1/3 2/4 Rare apical-aspect ofepithelial cell Adrenal  0/4^(d) 0/7 Esophagus 1/2 0/3 Single focus ofsuprabasal cell membrane staining, membrane rim pattern Stomach  0/4^(c)0/6 [Intracellular canaliculas of parietal cells positive, 3/4]^(c)Small intestine  0/4^(c) 0/7 Large intestine  0/4^(c) 0/6 Gallbladder nt0/3 Testes 0/4 0/5 [Weak positive rete testes, collecting duetules, 2/3;epididymis negative, 3] Prostate 0/1 0/4 [2 specimens includedperiurethral prostatic ducts, weak positive] Ovary 0/3 0/5 [oneinclusion cyst positive, numerous inclusion cysts negative] Endometrium,proliferative 0/2 0/7 Heart 0/1 0/2 Skeletal muscle 0/2 0/3 Thymus nt0/3 Spleen 0/2 0/3 Lymph node 0/2 0/3 Epidermis 0/2 0/5 Othernon-epithelial see comments Tissues consistently negative in numeroussections: mesothelium, rbc, wbc, lymphoreticular cells, fat, smoothmuscle, nerve, endothelium Placenta nt 0/5 [Focal squamous metaplasticamnion cell, rim pattern, 3/3] Chorion nt Footnotes: ^(a)( ) totalnumber inclusive of double label studies; ^(b)not tested; ^(c)stainingof wispy extracellular material; ^(d)unidentified spindle cells incapsule focally positive; ^(e)[ ] additional specificity not counted intable.

TABLE 2 Immunohistochemical detection of CA6 in gynecologic and relatedneoplasms/hyperplasias. Formalin-fixed, paraffin-embedded tissuesections were stained with mab DS6 as the primary antibody byavidin-biotin immunohistochemistry. #positive/ Tissue #tested Ovarian:Serous cystadenoma 3/3 Serous borderline tumor 5/5 Serous adenocarcinoma11/11  (18/18)^(a) Mucinous cystadenoma 0/2 Mucinous borderline tumor0/2 Mucinous adenocarcinoma 3/9 Endometroid, borderline 0/1 Endometrial:Clear cell adenocarcinoma 2/2 Papillary serous adenocarcinoma 3/3Endometroid adenocarcinoma 1/1 Benign polyp 0/1 Peritoneum: Mesothelioma1/6 Reactive mesothelial hyperplasia 0/1 Serous surface carcinoma ofperitoneum 2/2 (3/3) Footnotes: ^(a)( ) total number inclusive of doublelabel studies

TABLE 3 Expression of Tumor-Associated Antigen CA6 in GynecologicalNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS61 + 2 + 3 + Reactive Nonreactive (Cases with (Cases with (Cases with >Cases/ (Cases with < 1–9% 10–49% 50% Total 1% neoplastic neoplasticneoplastic Major Number neoplastic cells cells cells Patterns ofNeoplasm of Cases cells reactive) reactive) reactive) reactive)Reactivity^(I) Uterine Cervix Squamous Cell Carcinoma 10/14 4 4 3 3 Me,Cy Squamous Dysplasia 0/7 7 Endocervical 0/2 2 Adenocarcinoma in SituEndocervical 2/3 1 1 1 Lu, Me Adenocarcinoma Adenosquamous Carcinoma 2/22 Me, Cy Endometrium Papillary Serous 6/6 2 2 2 Lu, Me AdenocarcinomaEndometriod 14/16 2 7 4 3 Lu, Me Adenocarcinoma Clear Cell Carcinoma 8/10 2 1 7 Lu, Me, Cy Mucinous Adenocarcinoma 3/5 2 1 2 Me, CyMalignant Mixed Mullerian 10/18 8 5 1 4 Me, Cy Tumor, carcinomatouscomponent Choriocarcinoma 0/1 1 Molar Pregnancy 0/3 3 Uterus Low GradeEndometrial 0/3 3 Stroma Sarcoma Undifferentiated 0/1 1 EndometrialSarcoma Leiomyoma 0/7 7 Leimyosarcoma 0/6 6 Ovary Serous Cystadenoma 9/12 3 1 8 Lu Papillary Serous Borderline 10/10 1 2 7 Lu TumorPapillary Seroris 56/58 2 4 9 43 Lu, Me, Cy Adenocarcinoma PapillarySerous Carcinoma 14/14 1 13 Lu, Me, Cy Metastases Mucinous Cystadenoma 0/10 10 Mucinous Borderline 0/8 8 Tumor, Intestinal Mucinous Borderline2/2 1 1 Lu Tumor, Mullerian Mucinous Adenocarcinoma  0/10 10 Clear CellCarcinoma 1/8 7 1 Me Endometriod 5/8 3 3 1 1 Lu, Me AdenocarcinomaBenign Brenner Tumor 6/9 3 2 1 3 Me, Lu, Cy Borderline Brenner Tumor 1/11 Me, Cy Granulosa Cell Tumor  0/15 15 Fibroma/Thecoma  0/11 11 SertoliCell Tumor 0/1 1 Yolk Sac Tumor 0/2 2 Struma Ovarii 0/1 1 Dysgerminoma0/3 2 Vulva Squamous Cell Carcinoma 2/3 1 2 Me, Cy HidradenomaPapilliferum 2/2 2 Me Peritoneum Papillary Serous Surface 2/2 1 1 Lu, MeCarcinoma Malignant Mesothelioma of 0/2 2 peritoneum Endosalpingiosis1/1 1 Lu (nonneoplastic) Mesothelium (incidental 0/7 7 nonneoplastic)^(I)Abbreviations for Patterns of Immunohistochemical Reactivity: Lu =Luminal cell membrane, Me = Cell membrane, Cy = Cytoplasm.

TABLE 4 Expression of Tumor-Associated Antigen CA6 in EpithelialNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive Nonreactive 1 + 2 + 3 + Cases/ (Cases with < (Cases with (Caseswith (Cases with > Major Total 1% 1–9% 10–49% 50% Patterns of Numbercells cells cells cells CA6 Neoplasm of Cases reactive) reactive)reactive) reactive) Expression Breast Infiltrating Ductal Carcinoma24/29 5 3 8 13 Lu, Me, ICL Infiltrating Lobular  5/11 6 1 1 3 ICLCarcinoma Pleomorphic Lobular 1/2 1 1 ICL, Lu Carcinoma ColloidCarcinoma 6/7 1 3 3 Lu, Me Invasive Apocrine 1/1 1 ICL CarcinomaMedullary Carcinoma 1/1 I Lu, Me, ICL Tubular Carcinoma 1/1 1 Lu, ICLInvasive Papillary 1/1 1 Lu, Me Carcinoma Brain Meningioma  3/25 22 2 1Cy, Me Craniopharyngioma 1/2 1 1 Lu Gallbladder GallbladderAdenocarcinoma 3/7 4 1 2 Lu, Me Cystic Duct Adenocarcinoma 1/2 1 1 LuGastrointestinal Colonic Adenocarcinoma  3/21 18 2 1 Lu, Me MetastaticColon 0/3 3 Adenocarcinoma Mucinous Colon 0/6 6 Adenocarcinoma ColonicTubular Adenoma 0/1 1 Cloacogenic Carcinoma 1/3 2 1 Lu, Me DuodenalAdenocarcinoma 0/1 1 Esophageal Adenocarcinoma 1/4 3 1 Lu EsophagealSquamous 2/9 7 1 1 Me, Cy Carcinoma Gastric Adenocarcinoma  7/19 12 2 5Lu, Me Gastric Squamous 1/1 1 Me, Cy Carcinoma Kidney Clear CellCarcinoma 13/18 5 13 Me, Lu Papillary Carcinoma 3/5 2 1 1 1 Lu, MeGranular Carcinoma 5/8 3 2 3 Me Chromophobe Carcinoma 3/4 1 2 1 Lu, Me,Cy Oncocytoma 2/2 1 Lu, Me, Cy Cortical Adenoma 1/1 1 Lu, Me Wilms Tumor1/9 8 1 Lu in tubules Larynx Squamous Cell Carcinoma 10/27 17 8 1 1 Me,Cy Squamous Papilloma 1/1 1 Lu, Me Liver Hepatocellular Carcinoma 1/8 71 Lu, Me, Cy Lung Adenocarcinoma  8/12 4 4 1 3 Lu, Me Squamous CellCarcinoma  6/15 9 1 4 1 Me, Cy Adenosquamous Carcinoma 1/1 1 Me PancreasAdenocarcinoma 8/9 1 2 3 3 Lu, Me Pleura Malignant Mesothelioma 0/6 6 MeProstate Acinar Adenocarcinoma  4/13 9 3 1 Lu, Me Prostatic Duct 0/5 5Adenocarcinoma Salivary Gland Epithelial Neoplasms [See 21/90 69 12 6 3Fig.] Skin Epithelial Neoplasms [See 19/79 60 15 4 Fig.] TesticleTeratoma, epithelial elements 2/5 3 2 Lu Thyroid Follicular Adenoma 0/22 Follicular Carcinoma  1/17 16 1 Lu Hurthle Cell Adenoma 2/5 3 1 1 LuHurthle Cell Carcinoma 2/5 3 1 1 Lu Hyperplastic Hurthle Cell 1/1 1 LuNodule Insular Carcinoma 1/1 1 Lu, Me Papillary Carcinoma 16/27 11 9 7Lu, Me Thymus Thymoma  0/17 17 Lu, Me Thymic Carcinoma 0/1 1 SquamousCell Carcinoma 1/1 1 Lu, Me Urinary Bladder Transistional Cell Carcinoma18/21 3 8 5 5 Lu, Me Squamous Cell Carcinoma 0/1 1 Abbreviations: Lu -Luminal portion of cell membrane; Me - Cell membrane; Cy - Cytoplasm;ICL - Intracytoplasmic lumina.

TABLE 5 Expression of Tumor-Associated Antigen CA6 in Skin Neoplasms asImmunohistochemically Detected by Monoclonal Antibody DS6 Reactive 1 +2 + 3 + Cases/ Nonreactive (Cases with (Cases with (Cases with > MajorTotal (Cases with < 1–9% 10–49% 50% Patterns of Number 1% cells cellscells CA6 Neoplasm of Cases cells reactive) reactive) reactive)reactive) Expression Basal Cell Carcinoma  0/19 19 Chondroid Syringoma0/1 1 Cylindroma 0/3 3 Dermatofibroma 0/9 9 Dermatofibosarcoma 0/3 3Protuberans Eccrine Aerospiroma 11/13 2 10 1 Me, Lu Eccrine Spiradenoma1/7 6 1 Lu Ecerine Carcinoma 0/1 1 Hemangioma 0/8 8 Leiomyoma 0/8 8Leiomyosarcoma 0/1 Merkel Cell Carcinoma 1/2 1 Punctate Cy, MeNeurofibroma  0/13 13 Nevus Sebaceous 0/5 5 Pilomatricoma 0/1 1 Poroma3/6 3 2 1 Me, Lu Seborrheic Keratosis 0/1 1 Spiradenoma 1/7 6 1 LuSyringoma 0/2 2 Syringocystadenoma 3/3 1 2 Lu PapilliferumTrichepithelioma  0/10 10 Urticaria Pigmentosa 0/3 3 Xanthogranuloma 0/44 Angiofibroma 0/2 2 Angiosarcoma 0/2 2 Glomangioma 0/1 1Hemangiopericytome 0/2 2 Malignant 0/1 1 Hemangiopericytoma Blue Nevus0/5 5 Spitz Nevus 0/2 2 Malignant Melanoma  0/10 10 Abbreviations: Lu -Luminal portion of cell membrane; Me - Cell Membrane; Cy - Cytoplasm.

TABLE 6 Expression of Tumor-Associated Antigen CA6 in Salivary GlandNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive 1 + 2 + 3 + Cases/ Nonreactive (Cases with (Cases with (Caseswith > Major Total (Cases with < 1–9% 10–49% 50% Patterns of Number 1%cells cells cells CA6 Neoplasm of Cases cells reactive) reactive)reactive) reactive) Expression Acinic Cell Carcinoma 0/8 8 AdenoidCystic Carcinoma  7/14 7 5 1 1 Lu Basal Cell Adenoma 1/4 3 1 Lu BasalCell Carcinoma 0/1 1 Clear Cell Carcinoma 0/5 5 Canalicular Adenoma 0/11 Malignant Mixed Tumor 0/1 1 Mucoepidermoid Carcinoma 3/4 1 3 Lu, MePleomorphic Adenoma  4/30 26 4 Lu Polymorphous Low Grade 0/1 1 CarcinomaSalivary Duct Carcinoma 4/5 1 2 2 Lu, Me, Cy Warthin Tumor  2/16 14 2 LuAbbreviations: Lu - Luminal portion of cell membrane; Me - CellMembrane; Cy - Cytoplasm.

TABLE 7 Non-Epithelial Neoplasms Which Do Not Express Tumor-AssociatedAntigen CA6 as Studied Immunohistochemically with Monoclonal AntibodyDS6 TOTAL CASES SITE NEOPLASM STUDIED Adrenal Neuroblastoma & 21Ganglioneuroblastoma Pheochromocytoma 4 Breast Fibroadenoma 1 BrainGlioblastoma 40 Multiforme Anaplastic 5 Astrocytoma Oligodendroglioma 9Pilocytic 12 Astrocytoma Medulloblastoma 10 Ependymoma 15 Ganglion Cell4 Tumors Central 1 Neurocytoma Schwannoma 29 Hemanioblastoma 6 Gastro-Appendiceal 5 intestinal Carcinoid Tumor Appendiceal 1 AdenocarcinoidColonic Carcinoid 5 Tumor Gastric Carcinoid 6 GI Mesenchymal 17 TumorsLung Carcinoid Tumor 21 Small Cell 5 Carcinoma Large Cell 6Neuroendocrine Carcinoma Lymphoma Diffuse Small Cell 31 Lymphomas LargeCell 19 Lymphomas Follicular 36 Lmphomas Blastic 11 Lymphomas HodgkinsDisease 29 Multiple Myeloma 23 Langerhans Cell 8 Histiocytosis LeukemiasAcute 4 Lymphoblastic Leukemia Acute 5 Myelogenous Leukemia Chronic 5Myelogenous Leukemia Oral Ameloblastoma 6 Tissues Pancreas Islet CellTumor 6 Parathyroid Adenoma 8 Skin Blue Nevus 5 Spitz Nevus 2 SkinMalignant 10 Melanoma Dermatofibroma 9 Dermatofibosarcoma 4 ProtuberansHemangioma 8 Leiomyoma 8 Leiomyosarcoma 1 Neurofibroma 13 Urticaria 3Pigmentosa Xanthogranuloma 4 Angiofibroma 2 Angiosarcoma 2 Glomangioma 1Hemangiopericytoma 2 Malignant 1 Hemangiopericytoma Soft Angiosarcoma 1Tissue Alveolar Soft 1 Parts Sarcoma Chondrosarcoma 2 Fibrosarcoma 2Granular Cell 7 Tumor Hemangiopericytoma 1 Leiomyosarcoma 14 Liposarcoma4 Malignant 6 Fibrous Histiocytoma Malignant 6 Schwannoma Neurofibroma 1Osteosarcoma 2 Rhabdomyosarcoma 3 Undifferentiated 2 SarcomaParaganglioma 5 Primitive 3 Neuroectodermal Tumor TesticleChoriocarcinoma 4 Embryonal 16 Carcinoma Seminoma 18 Teratoma, Non- 5epithelial elements Yolk Sac Tumor 12 Thyroid Medullary 3 Carcinoma

TABLE 8 Non-Epithelial Neoplasms Which Express Tumor-Associated AntigenCA6 as Detected Immunohistochemically by Monoclonal Antibody DS6Reactive 1 + 2 + 3 + Cases/ Nonreactive (Cases with (Cases with (Caseswith > Total (Cases with < 1–9% 10–49% 50% Major Patterns Number 1%cells cells cells of CA6 Neoplasm of Cases cells reactive) reactive)reactive) reactive) Expression Merkel Cell Carcinoma 1/2 1 1 Membranous& cytoplasmic Pituitary Adenoma  2/21 19 2 Luminal in pseudorosettes;punctate in cytoplasm Small Intestinal Carcinoid 1/9 8 1 Luminal inTumor rosettes Synovial Sarcoma 1/3 2 1 Luminal and membranous

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A monoclonal antibody selected from thegroup consisting of a monoclonal antibody produced by the hybridoma cellline DS6 deposited with the American Type Culture Collection as depositnumber PTA-4449 and fragments of the foregoing which specifically bindthe epitope bound by a monoclonal antibody produced by the hybridomacell line DS6 deposited with the American Type Culture Collection asdeposit number PTA-4449.
 2. The monoclonal antibody according to claim 1coupled to a detectable group.
 3. The monoclonal antibody according toclaim 1 coupled to a detectable group, said detectable group selectedfrom the group consisting of radiolabels, enzyme labels, fluorescentlabels, metal atoms, and chemiluminescent labels.
 4. The monoclonalantibody according to claim 1 coupled to a solid support.
 5. Themonoclonal antibody according to claim 1 coupled to a solid support,said solid support selected from the group consisting of beads, plates,slides and microtiter plate wells.
 6. The monoclonal antibody accordingto claim 1 coupled to a therapeutic agent.
 7. The monoclonal antibodyaccording to claim 1 coupled to a therapeutic agent, said therapeuticagent selected from the group consisting of radioisotopes, cytotoxicagents, chemotherapeutic agents, metals, and chemiluminescent agents. 8.The monoclonal antibody according to claim 1, wherein the monoclonalantibody is lyophilized.
 9. The monoclonal antibody according to claim1, wherein the monoclonal antibody is provided in a pharmaceuticalformulation comprising a pharmaceutically acceptable carrier.
 10. Themonoclonal antibody according to claim 1, wherein the monoclonalantibody is a chimeric antibody.
 11. The monoclonal antibody accordingto claim 1, wherein the monoclonal antibody is a humanized antibody. 12.The monoclonal antibody according to claim 6, wherein the monoclonalantibody is provided in a pharmaceutical formulation comprising apharmaceutically acceptable carrier.